The use of a non-coding region of chloroplast DNA in phylogenetic studies of the subtribeSonchinae (Asteraceae:Lactuceae)

The systematic utility of sequences from a non-coding region of chloroplast DNA (cpDNA) betweenpsbA andtrnH^(GUG) was examined by assessing phylogenetic relationships in subtribeSonchinae (Asteraceae:Lactuceae). Primers constructed against highly conserved regions of tRNA genes were used for PCR amplification and sequencing. ThepsbA-trnH intergenic spacer contains several insertions and deletions (indels) inSonchinae with the length varying from 385 to 450 bp. Sequence divergence ranges from 0.00% to 7.54% withinSonchinae, with an average of 2.4%. Average sequence divergence inSonchus subg.Sonchus is 2.0%, while the mean for subg.Dendrosonchus and its close relatives in Macaronesia (the woodySonchus alliance) is 1.0%. Our results suggest that this region does not evolve rapidly enough to resolve relationships among closely related genera or insular endemics in theAsteraceae. The phylogenetic utility ofpsbA-trnH sequences of the non-coding cpDNA was compared to sequences from the ITS region of nuclear ribosomal DNA. The results suggest that ITS sequences evolve nearly four times faster thanpsbA-trnH intergenic spacer sequences. Furthermore, the ITS sequences provide more variable and phylogenetically informative sites and generate more highly resolved trees with more strongly supported clades, and thus are more suitable for phylogenetic comparisons at lower taxonomic levels than thepsbA-trnH intergenic chloroplast sequences.     

The architecture of the chloroplast psbA-trnH non-coding region in angiosperms

The psbA-trnH intergenic region is among the most variable regions in the angiosperm chloroplast genome. It is a popular tool for plant population genetics and species level phylogenetics and has been proposed as suitable for DNA barcoding studies. This region contains two parts differing in their evolutionary conservation: 1) the psbA 3′UTR (untranslated region) and 2) the psbA-trnH intergenic non-transcribed spacer. We compared the sequence and RNA secondary structure of the psbA 3′ UTR across angiosperms and found consensus motifs corresponding to the stem portions of the RNA stem-loop structures and a consensus TTAGTGTATA box. The psbA-trnH spacer exhibited patterns that can be explained by the independent evolution of large inversions in the psbA 3′UTR and mutational hot spots in the remaining portion of the psbA-trnH spacer. We conclude that a comparison of chloroplast UTRs across angiosperms offer clues to the identity of putative regulatory elements and information about selective constraints imposed on the chloroplast non-coding regions.     

Phylogeny of Carpinus and subfamily Coryloideae (Betulaceae) based on chloroplast and nuclear ribosomal sequence data

Phylogenetic studies were conducted for Carpinus and the subfamily Coryloideae (Betulaceae) using sequences of the chloroplast matK gene, the trnL-trnF region (trnL intron, and trnL [UAA] 3' exon-trnF [GAA] intergenic spacer) and the psbA-trnH intergenic spacer, and the nuclear ribosomal ITS regions. The combined analyses of the three chloroplast regions suggest that Coryloideae is monophyletic; Ostryopsis is sister to the Carpinus - Ostrya clade; Corylus is monophyletic and sister to the Ostrya - Carpinus - Ostryopsis clade; Ostrya is paraphyletic; and within Carpinus, species of sect. Carpinus from eastern Asia form a monophyletic group, whereas the positions of C. betulus from Europe and C. caroliniana from eastern North America are unresolved within the Carpinus clade. The cpDNA tree generated in this study is largely congruent with the previously published ITS results, but the ITS tree places Carpinus sect. Distegocarpus as sister to the Ostrya - Carpinus sect. Carpinus clade. Future work is needed to examine the relationships within the Ostrya - Carpinus clade, evaluate the generic status of Ostrya, and test the phylogenetic position of Ostryopsis.     

Phylogenetic relationships in Blumea (Asteraceae: Inuleae) as evidenced by molecular and morphological data

The present study, based on sequences of cpDNA (trnL-F & psbA-trnH) and nrDNA (ITS) and morphology, examined the evolutionary relationships in Blumea and its position among related genera. The results confirmed that the closest relatives of Blumea are Caesulia, Duhaldea and Pentanema p.p., and showed that the monotypic genera Blumeopsis and Merrittia are nested within Blumea. In Blumea s.l., two major, well-supported clades were recognised and a single species, the widespread Blumea balsamifera, that could not be placed with certainty relative to the two main clades. The two main clades differ in habit, ecology and distribution. The Blumea densiflora clade contains shrubs and subshrubs of evergreen forests, distributed from continental Asia to New Guinea and Polynesia, whereas the Blumea lacera clade is a widespread paleotropical group that comprises mostly annual, weedy herbs of open forests and fields.     

Hybridization drives speciation in Gagea (Liliaceae)

Hybridization seems to play an important role in speciation of Gagea Salisb., a genus which is characterised by polyploid taxa lines and in which diploids (2n = 24) appear only to be common in basal sections. Hybrid detection was applied utilising direct and cloning nrDNA ITS data (ITS1, 5.8S rDNA, ITS2) combined with neighbour and ribotype networks and discussed in connection with previously published cpDNA, morphological and karyological data of the authors. We have evidence of the hybrid origin of taxa within the section Gagea (G. pomeranica, G. megapolitana) and the monophyletic clade of sections Didymobulbos and Fistulosae (G. microfistulosa, G. polidorii, G. cf. bohemica). Morphologically and karyologically differentiated Gagea megapolitana and G. pomeranica, adapted to synanthropic habitats, represent both hybrids of G. pratensis × G. lutea. Gagea microfistulosa represents a hybrid of G. villosa × G. fragifera; Gagea polidorii could represent the reverse hybrid. G. glacialis is also closely related to the latter complex.     

基于ITS和trnL-trnF序列的草木樨种群遗传多样性研究

以白花草木樨(Melilotus alba)和黄花草木樨(Melilotus officinalis)18个地理种群植物为材料,用ITS序列和trnL-trnF序列研究了2种草木樨不同种群间的遗传多样性。结果表明:(1)trnL-trnF序列对位后长度为459bp,其中包括6个变异位点,6个简约信息位点,G+C含量为33.1%;ITS序列对位后长度为714bp,其中包括5个变异位点,3个简约信息位点,G+C含量为48.9%。(2)在基于trnL-trnF序列构建的系统发育树中,2种草木樨能够形成单系分支,说明trnL-trnF序列在草木樨中的鉴别能力较强。(3)单倍型多样性以及核苷酸多样性分析表明,黄花草木樨的遗传多样性高于白花草木樨。     

基于叶绿体DNA片段的槭属鸡爪槭组系统进化和生物地理学研究

东亚地区经历过复杂的地质事件及气候变化,针对东亚地区进行专科专属的植物区系及地理演化研究具有重要的意义。基于3个叶绿体片段（psbA-trnH、rpl16和trnL-trnF）,对主要分布于东亚地区的鸡爪槭组（section Palmata Pax）18个分类群的分化时间、祖先地理分布、物种扩散路径等进行了分析。叶绿体片段总长度为2,232bp,其中变异位点341个（15.2%）,有效信息位点231个（10.3%）;18个鸡爪槭组内物种（包含亚种）被高度支持为单系群（99/95/1.0）,且组内物种间的支持度相对较高;鸡爪槭组内出现物种分化的时间多集中于中新世时期,组内物种的多样性速率保持相对稳定;根据BioGeoBears分析得出,东亚大陆局部地区是该组物种的祖先分布区,且现今分布于岛屿的物种分布在不同分支。通过讨论与分析得出：我国西南地区可能是鸡爪槭组植物的起源地;东海陆桥隐没与抬升,直接影响着鸡爪槭组物种的迁移与扩散;分布于亚热带亚洲大陆的鸡爪槭组物种,存在多次往同纬度高海拔地区扩散的现象。该研究有助于对东亚地区植物生物地理历史的进一步了解。     

Phylogeny of Rhododendron section Vireya (Ericaceae) based on two non-coding regions of cpDNA

Section Vireya (Blume) Copel.f. is one of the most morphologically diverse groups of the genus Rhododendron. Vireyas have a unique distribution for the genus, being predominantly found throughout the Malesian Archipelago. The alpha taxonomy of section Vireya is relatively well understood and taxa are easily distinguished from other rhododendrons by their general appearance. Defining characteristics of the section the possession of seeds with tailed appendages at both ends, the twisting of capsule valves after opening, placentas that separate as thread-like structures from the central axis as the capsule opens are all subject to exceptions. Phylogenetic analyses of two cpDNA regions, psbA-trnH and trnT-trnL intergenic spacers are reported. The results of each analysis were generally congruent, with clades relating strongly to geographic areas. Section Vireya is monophyletic with the inclusion of at least one species of section Rhododendron. Only two of the seven subsections currently circumscribed are monophyletic: Malayovireya Sleumer and Siphonovireya Sleumer. ``Euvireya', all subsections excluding the paraphyletic subsection Pseudovireya, is monophyletic and includes two major clades, one restricted to eastern Malesia and the other to the western and middle Malesia.     

Phylogenetic relationships within Pappophoreae s.l. (Poaceae: Chloridoideae): Additional evidence based on ITS and trnL-F sequence data

Historically, Pappophoreae included the genera Cottea, Enneapogon, Kaokochloa, Pappophorum and Schmidtia. Some authors consider this tribe as a well-supported monophyletic group; while other evidences reveals Pappophoreae as polyphyletic, with Pappophorum separated from the rest of the tribe. When the latter happens, it can form a clade with Tridens flavus. Molecular phylogenetic analyses of the subfamily Chloridoideae have included few species of Pappophoreae; therefore, further research involving more representatives of this tribe is needed. With the aim of providing new evidence to help clarify the phylogenetic position of Pappophorum and its relationships with other genera of the tribe and the subfamily Chloridoideae, eight new sequences of ITS and trnL-F regions of Pappophoreae species were generated. These sequences were analyzed together with other available sequence data obtained from GenBank, using maximum parsimony and Bayesian inference, for individual (trnL-F or ITS) or combined trnL-F/ITS data sets. All analyses reveal that Pappophoreae is polyphyletic, with Pappophorum separated from the rest of the tribe forming a well-supported clade sister to Tridens flavus.     

Molecular and morphological analyses of EuropeanAconitum species (Ranunculaceae)

The systematics of the yellow-floweredAconitum lycoctonum species complex (Ranunculaceae) has long been considered difficult because of high morphological variability and hypothesized hybridization. To elucidate the systematic value of these taxa, and to study their phylogeny in a broader context, we studied all species of the subgenusLycoctonum from Europe and the Caucasus Mountains and the European species of the subgen.Aconitum. We sequenced the ITS region and the chloroplast intergenic spacerpsbA-trnH and conducted parsimony analyses. We also measured morphological characters from 19A. lycoctonum populations. The two subgenera in Europe are clearly separated but hybridization between subgenera might have taken place in the past. Sequence variation withinA. lycoctonum and among all taxa of subgenusLycoctonum was very low and probably the result of recent speciation, leading to one morphologically highly variable species in which color morphs could be seen as subspecies. There was high morphological variability within and among populations and the morphological characters have no value as systematic characters.     

ITS phylogeny of West Asian Heracleum species and related taxa of Umbelliferae–Tordylieae W.D.J.Koch, with notes on evolution of their psbA-trnH sequences

Summary Heracleum is a large and taxonomically complex genus of the Umbelliferae–Tordylieae. The phylogenetic relationships of West Asian Heracleum species and related taxa were explored using data from sequences of the internal transcribed spacer (ITS1 and ITS2) region of nuclear ribosomal DNA. The data set consists of 56 species, of which 47 were analyzed for the first time; it represents all subdivisions of the genus Heracleum, as well as some representatives of Pastinaca complex. Heracleum was shown to be a polyphyletic genus, as its species fall into two different clades, one of which comprises also Symphyoloma and Mandenovia. Section Pubescentia was confirmed, in contrast to the sections Villosa and Heracleum being polyphyletic. A separate position of the section Wendia was supported. H. marashicum was shown to be a member of a clade comprising Pastinaca and related genera. The sequences of chloroplast psbA-trnH intergenic spacer, the region recently proposed for DNA barcoding in plants, were also analyzed for 33 species, representing all principal clades within Heracleum and its relatives. They have been proven to be very similar and not suitable for DNA barcoding in this group. However, some sequence variation was revealed. This variation could be explained by the combination of such evolutionary events as inversion and duplication. It was shown that these events are rather common in Tordylieae and can occur independently in different lineages. The evolution patterns of psbA-trnH spacer are hypothesized.     

Phylogenetic patterns in the fleshy-fruited Myrtaceae – preliminary molecular evidence

A phylogenetic study of selected fleshy-fruited genera of the Myrtaceae was conducted using sequences from the ITS region of nuclear DNA and the psbA-trnH region of plastid DNA. Studies to date have suggested that the fleshy-fruited state has arisen on several occasions in the Myrtaceae. The previously accepted and predominantly Neotropical tribe Myrteae has traditionally been divided into three groups, the subtribes Myrtinae, Eugeniinae and Myrciinae. This subtribal arrangement is analysed in detail here for the first time. The monophyly of the tribe and subtribes are tested and relationships of the genera within them, in particular those of the Myrciinae and anomalous genera sometimes associated with it, are discussed. Combined analyses of these two DNA regions revealed 40 shortest trees, all of which resolve Myrteae (excluding the Acmena group) as monophyletic. Myrciinae appears to be monophyletic whereas Myrtinae and Eugeniinae appear polyphyletic. The phylogenetic positions and relationships of the anomalous genera Myrceugenia, Luma and Blepharocalyx are unclear, but Myrceugenia is never included within the Myrciinae s.str. A Myrciinae s.str. clade emerges within which Myrcia, Calyptranthes and Marlierea appear polyphyletic. Clades emerge, however, that may reflect some natural groupings within the subtribe.We thank David Simpson, Lazlo Csiba, Edith Kapinos and many others from Kew for invaluable advice and support. It would not have been possible to collect the Brazilian samples without the patience and careful guidance of Dr. Vinicius C. Souza, Fiorella F. Mazine (Universidade de São Paulo, ESALQ), Professor Gert Hatschbach, Joel M. de Silva (Museu Botânico Municipal, Curitiba) and many others from the ESA and MBM herbaria. Thanks also to Les Landrum, Andrew Salywon, Marcos Sobral and an anonymous reviewer for helpful comments at different stages of this work. British Airways are gratefully acknowledged for providing a flight to Brazil under their Community and Conservation programme.     

Origin and phylogeny of Oryza species with the CD genome based on multiple-gene sequence data

The CD genome species in the genus Oryza are endemic to Latin America, including O. alta, O. grandiglumis and O. latifolia. Origins and phylogenetic relationship of these species have long been in dispute and are still ambiguous due to their homogeneous genome type, similar morphological characteristics and overlapping distribution. In the present study, we sequenced two chloroplast fragments (matK and trnL-trnF) and portions of three nuclear genes (Adh1, Adh2 and GPA1) from sixteen accessions representing seven species with the C, CD, and E genomes, as well as one G genome species as the outgroup. Phylogenetic analyses using parsimony and distance methods strongly supported that the CD genome originated from a single hybridization event, and that the C genome species (O. officinalis or O. rhizomatis instead of O. eichingeri) served as the maternal parent while the E genome species (O. australiensis) was the paternal donor during the formation of CD genome. In addition, the consistent phylogenetic relationships among the CCDD species indicated that significant divergence existed between O. latifolia and the other two (O. alta and O. grandiglumis), which corroborated the suggestion of treating the latter two as a single species or as taxa within species.We thank Tao Sang of Michigan State University (East Lansing, USA) and Bao-rong Lu of Fudan University (Shanghai, China) for their encouragement and assistance. We are also grateful to the International Rice Research Institute (Manila, Philippines) for providing plant material for this study. This research was supported by the Chinese Academy of Sciences (kscxz-sw-101A), the National Natural Science Foundation of China (30025005) and the Program for Key International S & T Cooperation Project of P. R. China (2001CB711103).     

Evaluation of six candidate DNA barcoding loci in Ficus (Moraceae) of China

Ficus, with about 755 species, diverse habits and complicated co‐evolutionary history with fig wasps, is a notoriously difficult group in taxonomy. DNA barcoding is expected to bring light to the identification of Ficus but needs evaluation of candidate loci. Based on five plastid loci (rbcL, matK, trnH‐psbA, psbK‐psbI, atpF‐atpH) and a nuclear locus [internal transcribed spacer (ITS)], we calculated genetic distances and DNA barcoding gaps individually and in combination and constructed phylogenetic trees to test their ability to distinguish the species of the genus. A total of 228 samples representing 63 putative species in Ficus (Moraceae) of China were included in this study. The results demonstrated that ITS has the most variable sites, greater intra‐ and inter‐specific divergences, the highest species discrimination rate (72%) and higher primer universality among the single loci. It is followed by psbK‐psbI and trnH‐psbA with moderate variation and considerably lower species discrimination rates (about 19%), whereas matK, rbcL and atpF‐atpH could not effectively separate the species. Among the possible combinations of loci, ITS + trnH‐psbA performed best but only marginally improved species resolution over ITS alone (75% vs. 72%). Therefore, we recommend using ITS as a single DNA barcoding locus in Ficus.     

A taxonomic reassessment of the Vittiaceae (Hypnales, Bryopsida): evidence from phylogenetic analyses of combined chloroplast and nuclear sequence data

The Vittiaceae are a small family of aquatic mosses that are defined based on gametophytic traits whose interpretation has led to conflicting taxonomic arrangements. Phylogenetic analyses of two cpDNA regions, trnL-trnF and atpB-rbcL, indicate that Vittia is nested within the Amblystegiaceae s. str., suggesting that the family Vittiaceae should not be recognized. Platylomella lescurii appears nested within the Thuidiaceae/Leskeaceae. This suggests that the series of character states shared by Vittia and Platylomella, including a differentiated leaf border, short laminal cells, stiff stems, and a thick costa, are convergent features that arose independently in unrelated lineages of aquatic Hypnales. Within the Amblystegiaceae, phylogenetic analyses of the two cpDNA regions combined with ITS sequence data show that Hypnobartlettia, Vittia elimbata spec. nov., V. pachyloma, and V. salina, despite their strong morphological similarity to aquatic Amblystegium species, form a clade that is sister to the Drepanocladus/Pseudo-calliergon complex. This combined clade is unresolved at a polytomy that includes Amblystegium serpens and a clade including all the other Amblystegium species. The occurrence of A. serpens outside the strongly supported clade including other Amblystegium species suggests that A. serpens may be better accommodated in a distinct genus. Amblystegium serpens is the type species of Amblystegium and thus retains the name. The other species are accommodated in their own genus, Hygroamblystegium, including H. fluviatile, H. humile comb. nov., H. noterophyllum, H. tenax, and H. varium.     

Extensive length variation in the cpDNA trnT-trnF region of hemiparasitic Pedicularis and its phylogenetic implications

The cpDNA trnT-trnF region, a molecular marker widely used in the phylogenetic reconstruction at lower taxonomic levels, is relatively conserved in size and structure. In this region single length variation over 100 bp is much less common than small deletion for congeneric species of angiosperms. Here we examined evolutionary patterns of the trnT-trnF region in 43 species of Pedicularis, a species-rich genus with adaptive radiation. Four independent large deletions, varying from 203 to 297 bp in length, were detected from nine species of the genus, which might result from slipped-strand mispairing. These deletions occurred in different locations of the cpDNA region and in different clades of the phylogenetic tree, indicating that the deletion of large cpDNA fragments may be very frequent in the hemiparasitic lineage of the family Orobanchaceae. Parsimony analyses showed that section Cyathophora of Pedicularis, endemic to the Sino-Himalayan region, was a strongly supported monophyletic group. This section could have a recent origin followed by rapid radiation, considering that it is characterized by a large deletion in the trnT-trnF region and a relatively low interspecific sequence divergence.     

TrnL–trnF Intergenic Spacer and trnL Intron Define Major Clades Within Luzula and Juncus (Juncaceae): Importance of Structural Mutations

Seven hundred fifty-two to one thousand ninety-seven base pairs of the trnL intron and trnL–trnF intergenic spacer of the chloroplast DNA of 55 Juncaceae taxa (Juncus, Luzula, Rostkovia, and Oxychloë) was sequenced. Seventeen structural mutations (13 indels marked A to M, 3 parts of the trnF pseudogene, and insertion o within a pseudogene) within the chloroplast trnL–trnF region were examined as possible indicators for phylogenetic relationships in Juncaceae. Juncus trifidus (section Steirochloa) was clearly separated from the other taxa by two large (>80 bp) indels. The Southern Hemisphere clade was strongly supported by a unique insertion (334 bp) in the trnL intron. The monophyly of Luzula was supported by three small (<10 bp) indels in the trnL-F spacer. They were found in all 22 examined members that represent the taxonomic and geographical diversity of the genus Luzula. A tandemly duplicated tRNA pseudogene was found in the Juncus subgenus Juncus species and is supported by four small unique indels too. The acceptor stem and D-domain-encoding regions are separated by a unique 8-bp insertion. The T-domain and acceptor stem-encoding regions were not found in the pseudogene repeats. Only the Juncus sections Ozophyllum and Iridifolii contain the 5 acceptor stem, D-domain, and anticodon domain of the tRNAF encoding DNA. The structural mutations in the trnL intron and the trnL–trnF intergenic spacer are useful for phylogenetic reconstruction in the Juncaceae.